It is a colorless, odorless solid, highly soluble in water, and practically non-toxic (LD50 is 15 g/kg for rats).[7] Dissolved in water, it is neither acidic nor alkaline. The body uses it in many processes, most notably nitrogen excretion. The liver forms it by combining two ammonia molecules (NH3) with a carbon dioxide (CO2) molecule in the urea cycle. Urea is widely used in fertilizers as a source of nitrogen (N) and is an important raw material for the chemical industry.
In 1828, Friedrich Wöhlerdiscovered that urea can be produced from inorganic starting materials, which was an important conceptual milestone in chemistry. This showed for the first time that a substance previously known only as a byproduct of life could be synthesized in the laboratory without biological starting materials, thereby contradicting the widely held doctrine of vitalism, which stated that only living organisms could produce the chemicals of life.
Properties
Molecular and crystal structure
The structure of the molecule of urea is O=C(−NH2)2. The urea molecule is planar when in a solid crystal because of sp2 hybridization of the N orbitals.[8][9] It is non-planar with C2 symmetry when in the gas phase[10] or in aqueous solution,[9] with C–N–H and H–N–H bond angles that are intermediate between the trigonal planar angle of 120° and the tetrahedral angle of 109.5°. In solid urea, the oxygen center is engaged in two N–H–O hydrogen bonds. The resulting hydrogen-bond network is probably established at the cost of efficient molecular packing: The structure is quite open, the ribbons forming tunnels with square cross-section. The carbon in urea is described as sp2 hybridized, the C-N bonds have significant double bond character, and the carbonyl oxygen is relatively basic. Urea's high aqueous solubility reflects its ability to engage in extensive hydrogen bonding with water.
By virtue of its tendency to form porous frameworks, urea has the ability to trap many organic compounds. In these so-called clathrates, the organic "guest" molecules are held in channels formed by interpenetrating helices composed of hydrogen-bonded urea molecules. In this way, urea-clathrates have been well investigated for separations.[11]
Reactions
Structure of [Fe(urea)6]2+ showing intramolecular hydrogen bonds.[12] Color code: blue = N, red = O.
Urea is a weak base, with a pKb of 13.9.[5] When combined with strong acids, it undergoes protonation at oxygen to form uronium salts.[13][14] It is also a Lewis base, forming metal complexes of the type [M(urea)6]n+.[15]
In aqueous solution, urea slowly equilibrates with ammonium cyanate. This elimination reaction[18] cogenerates isocyanic acid, which can carbamylate proteins, in particular the N-terminal amino group, the side chain amino of lysine, and to a lesser extent the side chains of arginine and cysteine.[19][20] Each carbamylation event adds 43 daltons to the mass of the protein, which can be observed in protein mass spectrometery.[20] For this reason, pure urea solutions should be freshly prepared and used, as aged solutions may develop a significant concentration of cyanate (20 mM in 8 M urea).[20] Dissolving urea in ultrapure water followed by removing ions (i.e. cyanate) with a mixed-bed ion-exchange resin and storing that solution at 4 °C is a recommended preparation procedure.[21] However, cyanate will build back up to significant levels within a few days.[20] Alternatively, adding 25–50 mM ammonium chloride to a concentrated urea solution decreases formation of cyanate because of the common ion effect.[20][22]
Analysis
Urea is readily quantified by a number of different methods, such as the diacetyl monoxime colorimetric method, and the Berthelot reaction (after initial conversion of urea to ammonia via urease). These methods are amenable to high throughput instrumentation, such as automated flow injection analyzers[23] and 96-well micro-plate spectrophotometers.[24]
A plant in Bangladesh that produces urea fertilizer.
More than 90% of world industrial production of urea is destined for use as a nitrogen-release fertilizer.[17] Urea has the highest nitrogen content of all solid nitrogenous fertilizers in common use. Therefore, it has a low transportation cost per unit of nitrogen nutrient. The most common impurity of synthetic urea is biuret, which impairs plant growth. Urea breaks down in the soil to give ammonium ions (NH+4). The ammonium is taken up by the plant through its roots. In some soils, the ammonium is oxidized by bacteria to give nitrate (NO−3), which is also a nitrogen-rich plant nutrient. The loss of nitrogenous compounds to the atmosphere and runoff is wasteful and environmentally damaging so urea is sometimes modified to enhance the efficiency of its agricultural use. Techniques to make controlled-release fertilizers that slow the release of nitrogen include the encapsulation of urea in an inert sealant, and conversion of urea into derivatives such as urea-formaldehyde compounds, which degrade into ammonia at a pace matching plants' nutritional requirements.
When urea is used, a pre-reaction (hydrolysis) occurs to first convert it to ammonia:
CO(NH2)2 + H2O → 2 NH3 + CO2
Being a solid highly soluble in water (545 g/L at 25 °C),[2] urea is much easier and safer to handle and store than the more irritant, caustic and hazardous ammonia (NH3), so it is the reactant of choice. Trucks and cars using these catalytic converters need to carry a supply of diesel exhaust fluid, also sold as AdBlue, a solution of urea in water.
Laboratory uses
Urea in concentrations up to 10 M is a powerful proteindenaturant as it disrupts the noncovalent bonds in the proteins. This property can be exploited to increase the solubility of some proteins. A mixture of urea and choline chloride is used as a deep eutectic solvent (DES), a substance similar to ionic liquid. When used in a deep eutectic solvent, urea gradually denatures the proteins that are solubilized.[26]
Urea in concentrations up to 8 M can be used to make fixed brain tissue transparent to visible light while still preserving fluorescent signals from labeled cells. This allows for much deeper imaging of neuronal processes than previously obtainable using conventional one photon or two photon confocal microscopes.[27]
Urea has also been studied as a diuretic. It was first used by Dr. W. Friedrich in 1892.[32] In a 2010 study of ICU patients, urea was used to treat euvolemic hyponatremia and was found safe, inexpensive, and simple.[33]
The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea. It is used as a marker of renal function, though it is inferior to other markers such as creatinine because blood urea levels are influenced by other factors such as diet, dehydration,[35] and liver function.
Urea has also been studied as an excipient in Drug-coated Balloon (DCB) coating formulation to enhance local drug delivery to stenotic blood vessels.[36][37] Urea, when used as an excipient in small doses (~3 μg/mm2) to coat DCB surface was found to form crystals that increase drug transfer without adverse toxic effects on vascular endothelial cells.[38]
Urea labeled with carbon-14 or carbon-13 is used in the urea breath test, which is used to detect the presence of the bacterium Helicobacter pylori (H. pylori) in the stomach and duodenum of humans, associated with peptic ulcers. The test detects the characteristic enzyme urease, produced by H. pylori, by a reaction that produces ammonia from urea. This increases the pH (reduces the acidity) of the stomach environment around the bacteria. Similar bacteria species to H. pylori can be identified by the same test in animals such as apes, dogs, and cats (including big cats).
Miscellaneous uses
An ingredient in diesel exhaust fluid (DEF), which is 32.5% urea and 67.5% de-ionized water. DEF is sprayed into the exhaust stream of diesel vehicles to break down dangerous NOxemissions into harmless nitrogen and water.
A component of animal feed, providing a relatively cheap source of nitrogen to promote growth
A non-corroding alternative to rock salt for road de-icing.[39] It is often the main ingredient of pet friendly salt substitutes although it is less effective than traditional rock salt or calcium chloride.[40]
A main ingredient in hair removers such as Nair and Veet
As an additive to extend the working temperature and open time of hide glue
As a solubility-enhancing and moisture-retaining additive to dye baths for textile dyeing or printing[43]
As an optical parametric oscillator in nonlinear optics[44][45]
Physiology
Amino acids from ingested food (or produced from catabolism of muscle protein) that are used for the synthesis of proteins and other biological substances can be oxidized by the body as an alternative source of energy, yielding urea and carbon dioxide.[46] The oxidation pathway starts with the removal of the amino group by a transaminase; the amino group is then fed into the urea cycle. The first step in the conversion of amino acids into metabolic waste in the liver is removal of the alpha-amino nitrogen, which produces ammonia. Because ammonia is toxic, it is excreted immediately by fish, converted into uric acid by birds, and converted into urea by mammals.[47]
Ammonia (NH3) is a common byproduct of the metabolism of nitrogenous compounds. Ammonia is smaller, more volatile, and more mobile than urea. If allowed to accumulate, ammonia would raise the pH in cells to toxic levels. Therefore, many organisms convert ammonia to urea, even though this synthesis has a net energy cost. Being practically neutral and highly soluble in water, urea is a safe vehicle for the body to transport and excrete excess nitrogen.
Urea is synthesized in the body of many organisms as part of the urea cycle, either from the oxidation of amino acids or from ammonia. In this cycle, amino groups donated by ammonia and L-aspartate are converted to urea, while L-ornithine, citrulline, L-argininosuccinate, and L-arginine act as intermediates. Urea production occurs in the liver and is regulated by N-acetylglutamate. Urea is then dissolved into the blood (in the reference range of 2.5 to 6.7 mmol/L) and further transported and excreted by the kidney as a component of urine. In addition, a small amount of urea is excreted (along with sodium chloride and water) in sweat.
In water, the amine groups undergo slow displacement by water molecules, producing ammonia, ammonium ions, and bicarbonate ions. For this reason, old, stale urine has a stronger odor than fresh urine.
By action of the urea transporter 2, some of this reabsorbed urea eventually flows back into the thin descending limb of the tubule,[49] through the collecting ducts, and into the excreted urine. The body uses this mechanism, which is controlled by the antidiuretic hormone, to create hyperosmotic urine — i.e., urine with a higher concentration of dissolved substances than the blood plasma. This mechanism is important to prevent the loss of water, maintain blood pressure, and maintain a suitable concentration of sodium ions in the blood plasma.
The equivalent nitrogen content (in grams) of urea (in mmol) can be estimated by the conversion factor 0.028 g/mmol.[50] Furthermore, 1 gram of nitrogen is roughly equivalent to 6.25 grams of protein, and 1 gram of protein is roughly equivalent to 5 grams of muscle tissue. In situations such as muscle wasting, 1 mmol of excessive urea in the urine (as measured by urine volume in litres multiplied by urea concentration in mmol/L) roughly corresponds to a muscle loss of 0.67 gram.
Other species
In aquatic organisms the most common form of nitrogen waste is ammonia, whereas land-dwelling organisms convert the toxic ammonia to either urea or uric acid. Urea is found in the urine of mammals and amphibians, as well as some fish. Birds and saurian reptiles have a different form of nitrogen metabolism that requires less water, and leads to nitrogen excretion in the form of uric acid. Tadpoles excrete ammonia, but shift to urea production during metamorphosis. Despite the generalization above, the urea pathway has been documented not only in mammals and amphibians, but in many other organisms as well, including birds, invertebrates, insects, plants, yeast, fungi, and even microorganisms.[51]
Adverse effects
Urea can be irritating to skin, eyes, and the respiratory tract. Repeated or prolonged contact with urea in fertilizer form on the skin may cause dermatitis.[52]
High concentrations in the blood can be damaging. Ingestion of low concentrations of urea, such as are found in typical human urine, are not dangerous with additional water ingestion within a reasonable time-frame. Many animals (e.g. camels, rodents or dogs) have a much more concentrated urine which may contain a higher urea amount than normal human urine.
Urea can cause algal blooms to produce toxins, and its presence in the runoff from fertilized land may play a role in the increase of toxic blooms.[53]
The substance decomposes on heating above melting point, producing toxic gases, and reacts violently with strong oxidants, nitrites, inorganic chlorides, chlorites and perchlorates, causing fire and explosion.[54]
This was the first time an organic compound was artificially synthesized from inorganic starting materials, without the involvement of living organisms. The results of this experiment implicitly discredited vitalism, the theory that the chemicals of living organisms are fundamentally different from those of inanimate matter. This insight was important for the development of organic chemistry. His discovery prompted Wöhler to write triumphantly to Jöns Jakob Berzelius:
"I must tell you that I can make urea without the use of kidneys, either man or dog. Ammonium cyanate is urea."
In fact, his second sentence was incorrect. Ammonium cyanate[NH4]+[OCN]− and urea CO(NH2)2 are two different chemicals with the same empirical formulaCON2H4, which are in chemical equilibrium heavily favoring urea under standard conditions.[62] Regardless, with his discovery, Wöhler secured a place among the pioneers of organic chemistry.
Uremic frost was first described in 1865 by Harald Hirschsprung, the first Danish pediatrician in 1870 who also described the disease that carries his name in 1886. Uremic frost has become rare since the advent of dialysis. It is the classical pre-dialysis era description of crystallized urea deposits over the skin of patients with prolonged kidney failure and severe uremia.[63]
Historical preparation
Urea was first noticed by Herman Boerhaave in the early 18th century from evaporates of urine. In 1773, Hilaire Rouelle obtained crystals containing urea from human urine by evaporating it and treating it with alcohol in successive filtrations.[64] This method was aided by Carl Wilhelm Scheele's discovery that urine treated by concentrated nitric acid precipitated crystals. Antoine François, comte de Fourcroy and Louis Nicolas Vauquelin discovered in 1799 that the nitrated crystals were identical to Rouelle's substance and invented the term "urea."[65][66]Berzelius made further improvements to its purification[67] and finally William Prout, in 1817, succeeded in obtaining and determining the chemical composition of the pure substance.[68] In the evolved procedure, urea was precipitated as urea nitrate by adding strong nitric acid to urine. To purify the resulting crystals, they were dissolved in boiling water with charcoal and filtered. After cooling, pure crystals of urea nitrate form. To reconstitute the urea from the nitrate, the crystals are dissolved in warm water, and barium carbonate added. The water is then evaporated and anhydrous alcohol added to extract the urea. This solution is drained off and evaporated, leaving pure urea.
Laboratory preparation
Ureas in the more general sense can be accessed in the laboratory by reaction of phosgene with primary or secondary amines:
COCl2 + 4 RNH2 → (RNH)2CO + 2 [RNH3]+Cl−
These reactions proceed through an isocyanate intermediate. Non-symmetric ureas can be accessed by the reaction of primary or secondary amines with an isocyanate.
In 2020, worldwide production capacity was approximately 180 million tonnes.[69]
For use in industry, urea is produced from synthetic ammonia and carbon dioxide. As large quantities of carbon dioxide are produced during the ammonia manufacturing process as a byproduct of burning hydrocarbons to generate heat (predominantly natural gas, and less often petroleum derivatives or coal), urea production plants are almost always located adjacent to the site where the ammonia is manufactured.
Synthesis
Urea plant using ammonium carbamate briquettes, Fixed Nitrogen Research Laboratory, ca. 1930
The basic process, patented in 1922, is called the Bosch–Meiser urea process after its discoverers Carl Bosch and Wilhelm Meiser.[70] The process consists of two main equilibrium reactions, with incomplete conversion of the reactants. The first is carbamate formation: the fast exothermic reaction of liquid ammonia with gaseous carbon dioxide (CO2) at high temperature and pressure to form ammonium carbamate ([NH4]+[NH2COO]−):[17]
2 NH3 + CO2 ⇌ NH4CO2NH2(ΔH = −117 kJ/mol at 110 atm and 160 °C)[17][71]
The second is urea conversion: the slower endothermic decomposition of ammonium carbamate into urea and water:
The overall conversion of NH3 and CO2 to urea is exothermic, with the reaction heat from the first reaction driving the second. The conditions that favor urea formation (high temperature) have an unfavorable effect on the carbamate formation equilibrium. The process conditions are a compromise: the ill-effect on the first reaction of the high temperature (around 190 °C) needed for the second is compensated for by conducting the process under high pressure (140–175 bar), which favors the first reaction. Although it is necessary to compress gaseous carbon dioxide to this pressure, the ammonia is available from the ammonia production plant in liquid form, which can be pumped into the system much more economically. To allow the slow urea formation reaction time to reach equilibrium, a large reaction space is needed, so the synthesis reactor in a large urea plant tends to be a massive pressure vessel.
Reactant recycling
Because the urea conversion is incomplete, the urea must be separated from the unconverted reactants, including the ammonium carbamate. Various commercial urea processes are characterized by the conditions under which urea forms and the way that unconverted reactants are further processed.
Conventional recycle processes
In early "straight-through" urea plants, reactant recovery (the first step in "recycling") was done by letting down the system pressure to atmospheric to let the carbamate decompose back to ammonia and carbon dioxide. Originally, because it was not economic to recompress the ammonia and carbon dioxide for recycle, the ammonia at least would be used for the manufacture of other products such as ammonium nitrate or ammonium sulfate, and the carbon dioxide was usually wasted. Later process schemes made recycling unused ammonia and carbon dioxide practical. This was accomplished by the "total recycle process", developed in the 1940s to 1960s and now called the "conventional recycle process". It proceeds by depressurizing the reaction solution in stages (first to 18–25 bar and then to 2–5 bar) and passing it at each stage through a steam-heated carbamate decomposer, then recombining the resulting carbon dioxide and ammonia in a falling-film carbamate condenser and pumping the carbamate solution back into the urea reaction vessel.[17]
Stripping recycle process
The "conventional recycle process" for recovering and reusing the reactants has largely been supplanted by a stripping process, developed in the early 1960s by Stamicarbon in The Netherlands, that operates at or near the full pressure of the reaction vessel. It reduces the complexity of the multi-stage recycle scheme, and it reduces the amount of water recycled in the carbamate solution, which has an adverse effect on the equilibrium in the urea conversion reaction and thus on overall plant efficiency. Effectively all new urea plants use the stripper, and many total recycle urea plants have converted to a stripping process.[17][72]
In the conventional recycle processes, carbamate decomposition is promoted by reducing the overall pressure, which reduces the partial pressure of both ammonia and carbon dioxide, allowing these gasses to be separated from the urea product solution. The stripping process achieves a similar effect without lowering the overall pressure, by suppressing the partial pressure of just one of the reactants in order to promote carbamate decomposition. Instead of feeding carbon dioxide gas directly to the urea synthesis reactor with the ammonia, as in the conventional process, the stripping process first routes the carbon dioxide through the stripper. The stripper is a carbamate decomposer that provides a large amount of gas-liquid contact. This flushes out free ammonia, reducing its partial pressure over the liquid surface and carrying it directly to a carbamate condenser (also under full system pressure). From there, reconstituted ammonium carbamate liquor is passed to the urea production reactor. That eliminates the medium-pressure stage of the conventional recycle process.[17][72]
Side reactions
The three main side reactions that produce impurities have in common that they decompose urea.
Urea hydrolyzes back to ammonium carbamate in the hottest stages of the synthesis plant, especially in the stripper, so residence times in these stages are designed to be short.[17]
Biuret is formed when two molecules of urea combine with the loss of a molecule of ammonia.
2 NH2CONH2 → NH2CONHCONH2 + NH3
Normally this reaction is suppressed in the synthesis reactor by maintaining an excess of ammonia, but after the stripper, it occurs until the temperature is reduced.[17] Biuret is undesirable in urea fertilizer because it is toxic to crop plants to varying degrees,[73] but it is sometimes desirable as a nitrogen source when used in animal feed.[74]
This decomposition is at its worst when the urea solution is heated at low pressure, which happens when the solution is concentrated for prilling or granulation (see below). The reaction products mostly volatilize into the overhead vapours, and recombine when these condense to form urea again, which contaminates the process condensate.[17]
Corrosion
Ammonium carbamate solutions are highly corrosive to metallic construction materials – even to resistant forms of stainless steel – especially in the hottest parts of the synthesis plant such as the stripper. Historically corrosion has been minimized (although not eliminated) by continuous injection of a small amount of oxygen (as air) into the plant to establish and maintain a passive oxide layer on exposed stainless steel surfaces. Highly corrosion resistant materials have been introduced to reduce the need for passivation oxygen, such as specialized duplex stainless steels in the 1990s, and zirconium or zirconium-clad titanium tubing in the 2000s.[17]
Finishing
Urea can be produced in solid forms (prills, granules, pellets or crystals) or as solutions.
Solid forms
For its main use as a fertilizer urea is mostly marketed in solid form, either as prills or granules. Prills are solidified droplets, whose production predates satisfactory urea granulation processes. Prills can be produced more cheaply than granules, but the limited size of prills (up to about 2.1 mm in diameter), their low crushing strength, and the caking or crushing of prills during bulk storage and handling make them inferior to granules. Granules are produced by acretion onto urea seed particles by spraying liquid urea in a succession of layers. Formaldehyde is added during the production of both prills and granules in order to increase crushing strength and suppress caking. Other shaping techniques such as pastillization (depositing uniform-sized liquid droplets onto a cooling conveyor belt) are also used.[17]
Liquid forms
Solutions of urea and ammonium nitrate in water (UAN) are commonly used as a liquid fertilizer. In admixture, the combined solubility of ammonium nitrate and urea is so much higher than that of either component alone that it gives a stable solution with a total nitrogen content (32%) approaching that of solid ammonium nitrate (33.5%), though not, of course, that of urea itself (46%). UAN allows use of ammonium nitrate without the explosion hazard.[17] UAN accounts for 80% of the liquid fertilizers in the US.[75]
^ abNomenclature of Organic Chemistry : IUPAC Recommendations and Preferred Names 2013 (Blue Book). Cambridge: The Royal Society of Chemistry. 2014. pp. 416, 860–861. doi:10.1039/9781849733069-FP001. ISBN978-0-85404-182-4. The compound H2N-CO-NH2 has the retained name 'urea', which is the preferred IUPAC name, with locants N and N′, as shown above the structure below. The systematic name is 'carbonic diamide', (…).
^Loeser E, DelaCruz M, Madappalli V (9 June 2011). "Solubility of Urea in Acetonitrile–Water Mixtures and Liquid–Liquid Phase Separation of Urea-Saturated Acetonitrile–Water Mixtures". Journal of Chemical & Engineering Data. 56 (6): 2909–2913. doi:10.1021/je200122b.
^ abCalculated from 14−pKa. The value of pKa is given as 0.10 by the CRC Handbook of Chemistry and Physics, 49th edition (1968–1969). A value of 0.18 is given by Williams, R. (24 October 2001). "pKa Data"(PDF). Archived from the original(PDF) on 24 August 2003.
^ abIshida, Tateki; Rossky, Peter J.; Castner, Edward W. (2004). "A Theoretical Investigation of the Shape and Hydration Properties of Aqueous Urea: Evidence for Nonplanar Urea Geometry". The Journal of Physical Chemistry B. 108 (45): 17583–17590. doi:10.1021/jp0473218. ISSN1520-6106.
^Worsch, Detlev; Vögtle, Fritz (2002). "Separation of enantiomers by clathrate formation". Topics in Current Chemistry. Springer-Verlag. pp. 21–41. doi:10.1007/bfb0003835. ISBN3-540-17307-2.
^ abcSchaber, Peter M.; Colson, James; Higgins, Steven; Thielen, Daniel; Anspach, Bill; Brauer, Jonathan (2004). "Thermal decomposition (pyrolysis) of urea in an open reaction vessel". Thermochimica Acta. 424 (1–2): 131–142. Bibcode:2004TcAc..424..131S. doi:10.1016/j.tca.2004.05.018. ISSN0040-6031.
^ abcdeBurgess, Richard R.; Deutscher, Murray P. (2009). Guide to protein purification. San Diego, Calif: Academic Press/Elsevier. p. 819. ISBN978-0-12-374536-1. OCLC463300660.
^Duo et al., (1992). Can. J. Chem. Eng, 70, 1014–1020.
^Durand, Erwann; Lecomte, Jérôme; Baréa, Bruno; Piombo, Georges; Dubreucq, Éric; Villeneuve, Pierre (1 December 2012). "Evaluation of deep eutectic solvents as new media for Candida antarctica B lipase catalyzed reactions". Process Biochemistry. 47 (12). Elsevier: 2081–2089. doi:10.1016/j.procbio.2012.07.027. ISSN1359-5113..
^Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A (August 2011). "Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain". Nature Neuroscience. 14 (11): 1481–8. doi:10.1038/nn.2928. PMID21878933. S2CID28281721.
^
Boerhaave called urea "sal nativus urinæ" (the native, i.e., natural, salt of urine). See:
The first mention of urea is as "the essential salt of the human body" in: Peter Shaw and Ephraim Chambers, A New Method of Chemistry …, vol 2, (London, England: J. Osborn and T. Longman, 1727), page 193: Process LXXXVII.
Lindeboom, Gerrit A. Boerhaave and Great Britain …, (Leiden, Netherlands: E.J. Brill, 1974), page 51.
Backer, H. J. (1943) "Boerhaave's Ontdekking van het Ureum" (Boerhaave's discovery of urea), Nederlands Tijdschrift voor Geneeskunde (Dutch Journal of Medicine), 87 : 1274–1278 (in Dutch).
